This invention relates to a process for producing a racemic mixture of N-acyl-D,L-phenylalanine esters. More particularly, this invention provides an especially effective process for obtaining a racemic mixture of N--C.sub.1-9 acyl-D,L-phenylalanine ester which can be subsequently converted to both L-phenylalanine and D-phenylalanine by treatment with microbially derived serine proteinases.
Until recently, the resolution of racemic D,L-phenylalanine mixtures has followed three different routes of optical resolution in order to obtain one of the optically active antipodes. The first includes asymmetric hydrolysis of N-chloroacetyl-D,L-phenylalanine by the carboxypeptidase in pancrease. A second involves asymmetric hydrolysis of N-acetyl-D,L-phenylalanine by mold aminoacylase which process can be operated in a continuous fashion using a packed column. And, finally, a physico-chemical resolution based on preferential crystallization of isomers from supersaturated solution of acetyl-D,L-phenylalanine ammonium salt. In addition, there are several synthetic methods for production of L-phenylalanine. For example, conversion of L-tyrosine, treatment of synthetic phenylpyruvic acid with transaminase and enzymatic isomerization of synthetic D,L-phenylalanine by microorganisms. Kaneko et al, Synthetic Production and Utilization of Amino Acids, J. Wiley & Sons, pp. 171-179 (1974).
In Belgian Patent 855,051, there is taught a method and a composition for treatment of D,L-amino acids to resolve the racemic mixture and obtain L-isomer in which a supported aminoacylase is used to treat aqueous solutions of the D,L-amino acid. The support is a porous inorganic substrate of specified grain size, surface area, pore diameter and volume and is coated with a network polymer film, or carries a tertiary amine or quaternary ammonium salt group. Typical supports are titania, alumina or silica. The network polymer can be polyamino epoxides, polyamine-formaldehyde, phenol-formaldehyde mixtures and polymerized mixtures of vinyl monomers. The aminoacylases are enzymes of animal origin, such as pork kidney extracts or microorganism products, such as from Aspergillus, Lactobacillus arabinosus, Micrococcus glutamicus, and Pseudomonoas cruciviae. The process includes contacting the amino acids, such as N-acetyl-D,L-amino acid, with the complex of support/tertiary amine/polymer network/enzyme, for example, by passing through a packed column.
In U.S. Pat. No. 3,963,573, there is taught a process for producing optically pure N-acyl-L-methionine by subjecting an N-acyl-D,L-methionine ester to the action of a proteolytic enzyme selected from the group consisting of sulfhydryl proteinases and microbially derived serine proteinases and separating the resulting N-acyl-L-methionine. The art has recognized that certain proteolytic enzymes can be produced in a pure form, such as from Bacillus subtilis, Guntelberg Trav. Lab. Carlsberg, Ser. Chim. Vol. 29, p. 36 (1954). The proteolytic enzyme prepared from the strain of Bacillus subtilis was purified by crystallization and its physico-chemical properties were determined. The enzymatic properties were investigated insofar as optimum pH for milk coagulation, stability, degradation of casein, hydrolysis of hemoglobin, activators and inhibitors for the enzymes, the effect on ovalbumin and other characteristics. In the Journal of Biological Chemistry, Vol. 243, No. 7, pp. 1344-1348 (1968), Barel, examined the activity of Carlsberg and Novo subtilisins toward a number of N-acetylamino acid esters and amino acid esters. The enzymes were also compared with respect to their efficiency in catalyzing aminolysis reactions, their rates of inactivation by certain aromatic sulfonyl halides and the rates of deacylation of their N-trans-cinnamoyl derivatives. Although the enzymes were found qualitatively indistinguishable from the standpoint of substrate specificity, significant quantitative differences were observed. Thus, the microbially derived serine proteinases, for example, Novo and Carlsberg subtilisins exhibited varying of degrees of esterase activity on various N-acyl-L-amino acid esters.
In U.S. Pat. No. 4,262,092, incorporated herein by reference, there is disclosed a process for resolution of N-acyl-D,L-phenylalanine esters employing the activity of serine proteinases. According to the disclosure, optically pure N-acyl-D-phenylalanine ester is produced by reacting a racemic mixture of N-acyl-D,L-phenylalanine ester with a proteolytic enzyme and separating the resulting N-acyl-D-phenylalanine ester from the solution with subsequent removal of the N-acyl and ester groups to provide D-phenylalanine.
Subjecting N-acyl-D,L-phenylalanine ester to the action of such a proteinase provides a mixture of N-acyl-D-phenylalanine ester and N-acyl-L-phenylalanine. The N-acyl-L-phenylalanine can be readily separated from the mixture by conventional means, for example, by adjusting the pH of an aqueous mixture thereof and extracting with an organic solvent such as chloroform, ethyl acetate, butyl acetate, methylene chloride and the like. The isolated N-acyl-L-phenylalanine can then be hydrolyzed to yield L-phenylalanine. The N-acyl-D-phenylalanine ester can be converted to D-phenylalanine by known simple hydrolysis procedures such as dissolving the ester in 2 N HBr and heating for a time at 80.degree.-100.degree. C. The preparation of amino acids by subjecting a racemic mixture of N-acyl-D-phenylalanine ester and N-acyl-L-phenylalanine ester to the action of a proteolytic enzyme provides the economic benefits of producing high purity material in high yields at a rapid rate while using a readily available and inexpensive material, such as enzyme. Accordingly, if a simple one-step method were available for producing a racemic mixture of N-acyl-D,L-phenylalanine esters which could be inexpensively treated with proteolytic enzyme in accordance with the foregoing procedure to produce both L-phenylalanine and D-phenylalanine, the overall process would provide an even more attractive route to phenylalanine.
In accordance with the present invention, such a process now has been discovered and involves the catalytic hydrogenation of the azlactone of N-acyl-acetaminocinnamic acid over a nickel hydrogenation catalyst to produce a racemic mixture of N-acyl-D,L-phenylalanine ester.
While there are several methods available for converting azlactones to the corresponding acylamino acids or amino acids, no process has been disclosed or is available insofar as the Applicant is aware for converting an azlactone to a racemic mixture of N-acyl-D,L-phenylalanine ester. For example, in the Journal of Organic Chemistry, Vol. 37, No. 18, pp. 2916-2918 (1972), Badshah, Khan and Kidwai report that there are three general methods, employing reduction and hydrolysis, for the conversion of azlactones to the corresponding acylamino acids or amino acids. Reduction can be effected with sodium or sodium amalgam in water or ethanol, with hydriodic acid and red phosphorus in acetic acid or acetic anhydride, or catalytically over platinum or palladium in the presence of hydrogen. Though most amino acids, excepting tryptophane, have been synthesized by treatment with hydriodic acid and red phosphorus, the method using sodium or amalgam is not of wide applicability. Catalytic reduction has been less favored owing to the high cost of platinum and palladium which becomes a factor in large scale laboratory preparations and resistance of azlactones to hydrogenation which requires their initial hydrolysis to the unsaturated acylamino acids. In an attempt to improve upon the catalytic hydrogenation method for preparing amino acids, Badshah, Khan and Kidwai devised a method for preparing amino acids from azlactones utilizing a reductive hydrogenation of a suspension of azlactone in alcoholic ammonia of Raney nickel at elevated hydrogenation pressure and room temperature. The resulting acylamino acid amide is then hydrolyzed either to acylamino acid or to the desired amino acid by mild or stringent treatment with acid or alkali.
In Homogeneous Catalysis, Vol. 2, pp. 274-282, Knowles, Sabacky and Vineyard report the preparation of .alpha.-amino acids by an asymmetric hydrogenation of azlactone using a rhodiumchiral phosphine catalyst.